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- EMDB-15698: Cryo-EM structure of Cas12k-sgRNA binary complex (type V-K CRISPR... -

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Basic information

Entry
Database: EMDB / ID: EMD-15698
TitleCryo-EM structure of Cas12k-sgRNA binary complex (type V-K CRISPR-associated transposon)
Map data
Sample
  • Complex: Binary complex of ShCas12k bound to sgRNA.
    • Protein or peptide: Cas12k
    • RNA: sgRNA
  • Ligand: water
KeywordsCRISPR / complex / transposition / DNA BINDING PROTEIN
Function / homology: / Cas12k
Function and homology information
Biological speciesScytonema hofmannii (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.87 Å
AuthorsTenjo-Castano F / Sofos N / Stella S / Molina R / Pape T / Lopez-Mendez B / Stutzke LS / Temperini P / Montoya G
Funding support Denmark, 4 items
OrganizationGrant numberCountry
Novo Nordisk FoundationNNF0024386 Denmark
Novo Nordisk FoundationNNF14CC0001 Denmark
Novo Nordisk FoundationNNF17SA0030214 Denmark
Novo Nordisk FoundationNNF18OC0055061 Denmark
CitationJournal: Mol Cell / Year: 2024
Title: Conformational landscape of the type V-K CRISPR-associated transposon integration assembly.
Authors: Francisco Tenjo-Castaño / Nicholas Sofos / Luisa S Stutzke / Piero Temperini / Anders Fuglsang / Tillmann Pape / Pablo Mesa / Guillermo Montoya /
Abstract: CRISPR-associated transposons (CASTs) are mobile genetic elements that co-opt CRISPR-Cas systems for RNA-guided DNA transposition. CASTs integrate large DNA cargos into the attachment (att) site ...CRISPR-associated transposons (CASTs) are mobile genetic elements that co-opt CRISPR-Cas systems for RNA-guided DNA transposition. CASTs integrate large DNA cargos into the attachment (att) site independently of homology-directed repair and thus hold promise for eukaryotic genome engineering. However, the functional diversity and complexity of CASTs hinder an understanding of their mechanisms. Here, we present the high-resolution cryoelectron microscopy (cryo-EM) structure of the reconstituted ∼1 MDa post-transposition complex of the type V-K CAST, together with different assembly intermediates and diverse TnsC filament lengths, thus enabling the recapitulation of the integration complex formation. The results of mutagenesis experiments probing the roles of specific residues and TnsB-binding sites show that transposition activity can be enhanced and suggest that the distance between the PAM and att sites is determined by the lengths of the TnsB C terminus and the TnsC filament. This singular model of RNA-guided transposition provides a foundation for repurposing the system for genome-editing applications.
History
DepositionAug 31, 2022-
Header (metadata) releaseApr 10, 2024-
Map releaseApr 10, 2024-
UpdateOct 30, 2024-
Current statusOct 30, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15698.png

Downloads & links

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Map

FileDownload / File: emd_15698.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 384 pix.
= 319.488 Å		0.83 Å/pix.
x 384 pix.
= 319.488 Å		0.83 Å/pix.
x 384 pix.
= 319.488 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.832 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.78466034 - 1.5510019
Average (Standard dev.)0.00009390345 (±0.02422348)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 319.488 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15698_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Post-processed by DeepEMhancer. Used for model building.

Fileemd_15698_additional_1.map
AnnotationPost-processed by DeepEMhancer. Used for model building.
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_15698_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_15698_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : Binary complex of ShCas12k bound to sgRNA.

EntireName: Binary complex of ShCas12k bound to sgRNA.
Components
  • Complex: Binary complex of ShCas12k bound to sgRNA.
    • Protein or peptide: Cas12k
    • RNA: sgRNA
  • Ligand: water

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Supramolecule #1: Binary complex of ShCas12k bound to sgRNA.

SupramoleculeName: Binary complex of ShCas12k bound to sgRNA. / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 159 KDa

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Macromolecule #1: Cas12k

MacromoleculeName: Cas12k / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 74.738258 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSQITIQARL ISFESNRQQL WKLMADLNTP LINELLCQLG QHPDFEKWQQ KGKLPSTVVS QLCQPLKTDP RFAGQPSRLY MSAIHIVDY IYKSWLAIQK RLQQQLDGKT RWLEMLNSDA ELVELSGDTL EAIRVKAAEI LAIAMPASES DSASPKGKKG K KEKKPSSS ...String:
MSQITIQARL ISFESNRQQL WKLMADLNTP LINELLCQLG QHPDFEKWQQ KGKLPSTVVS QLCQPLKTDP RFAGQPSRLY MSAIHIVDY IYKSWLAIQK RLQQQLDGKT RWLEMLNSDA ELVELSGDTL EAIRVKAAEI LAIAMPASES DSASPKGKKG K KEKKPSSS SPKRSLSKTL FDAYQETEDI KSRSAISYLL KNGCKLTDKE EDSEKFAKRR RQVEIQIQRL TEKLISRMPK GR DLTNAKW LETLLTATTT VAEDNAQAKR WQDILLTRSS SLPFPLVFET NEDMVWSKNQ KGRLCVHFNG LSDLIFEVYC GNR QLHWFQ RFLEDQQTKR KSKNQHSSGL FTLRNGHLVW LEGEGKGEPW NLHHLTLYCC VDNRLWTEEG TEIVRQEKAD EITK FITNM KKKSDLSDTQ QALIQRKQST LTRINNSFER PSQPLYQGQS HILVGVSLGL EKPATVAVVD AIANKVLAYR SIKQL LGDN YELLNRQRRQ QQYLSHERHK AQKNFSPNQF GASELGQHID RLLAKAIVAL ARTYKAGSIV LPKLGDMREV VQSEIQ AIA EQKFPGYIEG QQKYAKQYRV NVHRWSYGRL IQSIQSKAAQ TGIVIEEGKQ PIRGSPHDKA KELALSAYNL RLTRRSG SE FELENLYFQ

UniProtKB: Cas12k

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Macromolecule #2: sgRNA

MacromoleculeName: sgRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 84.006516 KDa
SequenceString: GGAUAUUAAU AGCGCCGCAA UUCAUGCUGC UUGCAGCCUC UGAAUUUUGU UAAAUGAGGG UUAGUUUGAC UGUAUAAAUA CAGUCUUGC UUUCUGACCC UGGUAGCUGC UCACCCUGAU GCUGCUGUCA AUAGACAGGA UAGGUGCGCU CCCAGCAAUA A GGGCGCGG ...String:
GGAUAUUAAU AGCGCCGCAA UUCAUGCUGC UUGCAGCCUC UGAAUUUUGU UAAAUGAGGG UUAGUUUGAC UGUAUAAAUA CAGUCUUGC UUUCUGACCC UGGUAGCUGC UCACCCUGAU GCUGCUGUCA AUAGACAGGA UAGGUGCGCU CCCAGCAAUA A GGGCGCGG AUGUACUGCU GUAGUGGCUA CUGAAUCACC CCCGAUCAAG GGGGAACCCU CCAAAAGGUG GGUUGAAAGG AG AAGUCAU UUAAUAAGGC CACU

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 2 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration8.0 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
300.0 mMKClsodium chloride
30.0 mMHEPES2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid
5.0 mMMgCl2magnesium chloride
1.0 mMTCEPTris(2-carboxyethyl)phosphine
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 3 s blotting, 4 degrees celcius.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 5049 / Average exposure time: 40.0 sec. / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.3000000000000003 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 96000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2228644
Startup modelType of model: NONE
Details: Ab initio reconstruction in cryoSPARC from the same data.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.87 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Number images used: 180258
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final 3D classificationNumber classes: 10 / Avg.num./class: 126000 / Software - Name: cryoSPARC (ver. 3.3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-8axb:
Cryo-EM structure of Cas12k-sgRNA binary complex (type V-K CRISPR-associated transposon)

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